42nd Annual Meeting of the Society of Toxicology. Salt Lake City, UT. March 9-13, 2003.

Temporal effects of AH receptor ligands on CH12LX murine B-cell lymphoma gene expression: cDNA microarray analysis, real-time PCR verification and bioinformatic assessment.

Fielden MR, Boverhof DR, Zacharewski TR.

Department of Biochemistry & Molecular Biology, National Food Safety & Toxicology Center, and Institute for Environmental Toxicology, Michigan State University, East Lansing, MI.

The B-cell, a major component of humoral immunity, is a sensitive target for the immunotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) possibly by rendering B-cells less responsive to antigen or mitogen stimulation. The potential mechanisms of TCDD action on B-cells were examined in murine B-cell lymphoma cells (CH12LX) treated with 3 nM TCDD or DMSO vehicle for 0, 2, 4, 6, 8, 12 and 24 hrs using a sequence verified cDNA microarray representing 3068 genes/ESTs. Quantitative real-time PCR was used to verify selected microarray results and statistical significance was determined using the t-test (p<0.05). Cyp1a1 and Cyp1a2 displayed characteristic induction profiles with maximum induction of 300- and 6-fold, respectively, at 4 hrs. Data was imported into GeneSpring for further analysis and gene clustering revealed a group of early induced genes that clustered with Cyp1a1 as well as a cluster of late down regulated immunologically relevant genes. For these responsive genes, a 3000 bp region upstream of the transcriptional start site was extracted from the UCSC Genome Browser database. Putative DREs were identified within the promoter regions of the “Cyp1a1 like” cluster which had position weight matrix scores comparable to 15 bona fide functional DREs suggesting that these genes may be regulated by the AHR. Induction by structurally diverse AHR ligands further implicated regulation of these genes by the AHR. Conversely, genes in the late down regulated cluster displayed a low incidence of putative DREs, suggesting secondary regulation. These genes are involved in mitogen-stimulated signaling pathways and therefore, may play a role in the compromised responsiveness of B-cells following treatment with TCDD and related ligands.